摘要
背景:间充质干细胞移植后在体内的归巢能力,会随着供体细胞在体外的培养传代而下降,从而严重影响了间充质干细胞对靶组织的修复作用。目的:探讨体外扩增传代对人骨髓来源间充质干细胞归巢相关因子的影响。方法:应用Ficoll密度梯度离心法将间充质干细胞自骨髓中分离出来,利用其贴壁生长的特性加以纯化,在常规培养条件下培养至第7代,观察原代及第3,5,7代细胞的形态学特点。采用MTT法检测第3,5,7代间充质干细胞的生长增殖特性,并绘制生长曲线。Real-time PCR法检测第3,5,7代间充质干细胞中归巢相关因子CXCR4、CXCR6、CXCL12(SDF-1)、CD44的表达,以2-△△Ct计算各代细胞目的基因的相对表达量,比较不同代次间充质干细胞中各细胞归巢因子表达的差异。结果与结论:采用密度梯度离心法自骨髓获得单个核细胞,将其贴壁培养传至第3代,可获得纯度较高的间充质干细胞。其在体外的增殖能力较强,但随着传代扩增次数增加,形态从细长梭形逐渐缩短变宽,增殖速率、总体扩增倍数以及CXCR4、CXCR6、CXCL12、CD44等细胞归巢因子的表达亦随传代培养而呈下降趋势。结果显示,间充质干细胞的归巢能力随扩增传代而逐渐下降,其机制可能与CXCR4、CXCR6、CXCL12、CD44等归巢相关因子在间充质干细胞中的表达水平下降有关。
BACKGROUND：The homing ability of mesenchymal stem cells after transplantation can decrease along with culture and passage in vitro. And the decline of homing abilities can further influence the implantation of mesenchymal stem cells in the target tissue, thus seriously affecting the repair effect.
OBJECTIVE：To investigate the effect and its related mechanisms by which in vitro culture and passage affect the homing ability of mesenchymal stem cells.
METHODS：Mesenchymal stem cells were isolated from the bone marrow using Ficol density gradient centrifugation, and then purified using adhesion method. The mesenchymal stem cells were cultured into the seventh generations with the normal cultural condition, and the morphological features of the 3rd, 5th and 7th generations of mesenchymal stem cells were observed. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide chromatometry was used to detect the growth feature of the 3rd, 5th and 7th generations of mesenchymal stem cells, and the growth curve was drawn. Real-time PCR was used to detect the expression of CXCR4, CXCR6, CXCL12, CD44 in the 3rd, 5th and 7th generations of mesenchymal stem cells, 2-△△Ct was calculated to get the relative value of each target gene, and the differences in expression of CXCR4, CXCR6, CXCL12, CD44 between different generations were compared.
RESULTS AND CONCLUSION：Mononuclear cells could be obtained from the bone marrow by using Ficol density gradient centrifugation. High-purity mesenchymal stem cells could be obtained using adherent method. The ability of in vitro growing was strong, but fol owing the passage, the cellmorphology became wider and shorter. And the proliferation rate, the overal proliferated multiple and the expression of homing related factors decreased fol owing the passage. The expression of CXCR4, CXCR6, CXCL12 and CD44 of mesenchymal stem cells decreased fol owing the passage. The homing ability of mesenchymal stem cells was decreased fol owing the passage, and may be relevant with the lower expression of CXCR4, CXCR6, CXCL12 and CD44 in cultured mesenchymal stem cells.
出处
《中国组织工程研究》
CAS
CSCD
2013年第40期7102-7109,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金面上项目(81270603)
天津市中青年骨干创新人才培养计划资助项目~~
